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Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton.

Sagara J, Arata T, Taniguchi S - PLoS ONE (2009)

Bottom Line: PP1-binding deficient mutants strongly induced cell retraction.Long and branched cytoplasmic processes were developed during the cell retraction.These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan. sagaraj@shinshu-u.ac.jp

ABSTRACT
Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

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Distribution of scapinin and actin in Hela cells.(A) Hela cells grown on glass coverslips were cultured in the presence (Tet+) or absence (Tet−) of 0.1 µg/ml tetracycline and were then fixed at 20 hours. After permeabilization, the distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively. Scapinin and actin are colocalized (arrows). (B) Confocal microscopic observation. Hela cells were cultured in the presence of 0.1 µg/ml tetracycline for 20 hours and were then stained with anti-scapinin antibody (green) and rhodamine-phalloidin (red) as in (A). Scapinin and actin are colocalized (arrows). Bar: 20 µm. (C) Absence of scapinin in actin stress fibers. Hela cells grown on a glass coverslip were cultured in the presence of 0.1 µg/ml tetracycline and were then fixed at 8 hours. The distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively as (A). There are four cells; two cells express low levels of scapinin (asterisks), and the other two cells express high levels of scapinin (arrow heads). Bar: 20 µm.
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pone-0004247-g005: Distribution of scapinin and actin in Hela cells.(A) Hela cells grown on glass coverslips were cultured in the presence (Tet+) or absence (Tet−) of 0.1 µg/ml tetracycline and were then fixed at 20 hours. After permeabilization, the distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively. Scapinin and actin are colocalized (arrows). (B) Confocal microscopic observation. Hela cells were cultured in the presence of 0.1 µg/ml tetracycline for 20 hours and were then stained with anti-scapinin antibody (green) and rhodamine-phalloidin (red) as in (A). Scapinin and actin are colocalized (arrows). Bar: 20 µm. (C) Absence of scapinin in actin stress fibers. Hela cells grown on a glass coverslip were cultured in the presence of 0.1 µg/ml tetracycline and were then fixed at 8 hours. The distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively as (A). There are four cells; two cells express low levels of scapinin (asterisks), and the other two cells express high levels of scapinin (arrow heads). Bar: 20 µm.

Mentions: Immunostaining showed that scapinin and actin were mostly colocalized at the edges of spreading cells (Fig. 5A). Confocal microscopic observation also showed colocalization of scapinin and actin (Fig. 5B). Shortly after tetracycline-treatment, when scapinin expression levels were low, actin stress fibers were seen in some cells, but as scapinin expression levels were increased, the number of actin stress fibers was reduced (Fig. 5C). In addition, scapinin staining was not seen in actin stress fibers (Fig. 5C). These results suggest that scapinin promotes cell spreading and motility through reorganization of the actin cytoskeleton.


Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton.

Sagara J, Arata T, Taniguchi S - PLoS ONE (2009)

Distribution of scapinin and actin in Hela cells.(A) Hela cells grown on glass coverslips were cultured in the presence (Tet+) or absence (Tet−) of 0.1 µg/ml tetracycline and were then fixed at 20 hours. After permeabilization, the distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively. Scapinin and actin are colocalized (arrows). (B) Confocal microscopic observation. Hela cells were cultured in the presence of 0.1 µg/ml tetracycline for 20 hours and were then stained with anti-scapinin antibody (green) and rhodamine-phalloidin (red) as in (A). Scapinin and actin are colocalized (arrows). Bar: 20 µm. (C) Absence of scapinin in actin stress fibers. Hela cells grown on a glass coverslip were cultured in the presence of 0.1 µg/ml tetracycline and were then fixed at 8 hours. The distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively as (A). There are four cells; two cells express low levels of scapinin (asterisks), and the other two cells express high levels of scapinin (arrow heads). Bar: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2626280&req=5

pone-0004247-g005: Distribution of scapinin and actin in Hela cells.(A) Hela cells grown on glass coverslips were cultured in the presence (Tet+) or absence (Tet−) of 0.1 µg/ml tetracycline and were then fixed at 20 hours. After permeabilization, the distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively. Scapinin and actin are colocalized (arrows). (B) Confocal microscopic observation. Hela cells were cultured in the presence of 0.1 µg/ml tetracycline for 20 hours and were then stained with anti-scapinin antibody (green) and rhodamine-phalloidin (red) as in (A). Scapinin and actin are colocalized (arrows). Bar: 20 µm. (C) Absence of scapinin in actin stress fibers. Hela cells grown on a glass coverslip were cultured in the presence of 0.1 µg/ml tetracycline and were then fixed at 8 hours. The distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively as (A). There are four cells; two cells express low levels of scapinin (asterisks), and the other two cells express high levels of scapinin (arrow heads). Bar: 20 µm.
Mentions: Immunostaining showed that scapinin and actin were mostly colocalized at the edges of spreading cells (Fig. 5A). Confocal microscopic observation also showed colocalization of scapinin and actin (Fig. 5B). Shortly after tetracycline-treatment, when scapinin expression levels were low, actin stress fibers were seen in some cells, but as scapinin expression levels were increased, the number of actin stress fibers was reduced (Fig. 5C). In addition, scapinin staining was not seen in actin stress fibers (Fig. 5C). These results suggest that scapinin promotes cell spreading and motility through reorganization of the actin cytoskeleton.

Bottom Line: PP1-binding deficient mutants strongly induced cell retraction.Long and branched cytoplasmic processes were developed during the cell retraction.These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan. sagaraj@shinshu-u.ac.jp

ABSTRACT
Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

Show MeSH
Related in: MedlinePlus